Adhesion of cells to their surrounding extracellular matrix (ECM) in vivo regulates their morphology, proliferation, migration, survival and differentiation. Adams et al. (1993) Development 117:1183-1198; Ashkenas et al. (1996) Dev. Biol. 180:433-444; Giancotti (1997) Curr. Opin. Cell. Biol. 9:691-700; Howe et al. (1998) Curr Opin. Cell Biol. 10:220-231. In vitro, the interactions of cells with ECM molecules such as fibronectin result in cell attachment, spreading and the assembly of focal adhesions and actin stress fibers. Burridge et al. (1996) Ann. Rev. Cell Dev. Biol. 12:463-518. Focal adhesions are macromolecular complexes made up of transmembrane adhesion receptors and intracellular proteins with structural and signaling functions. Burridge et al., supra; Clark et al. (1995) Science 268:233-239.
Two independent adhesion receptor-mediated signals are required for the assembly of these macromolecular complexes when cells are plated on fibronectin. One signal is mediated through integrins and involves the RGD-containing cell-binding domain of fibronectin. Hynes (1992) Cell 69:11-25; Clark et al. (1995) Science 268:233-239. The second signal is mediated through cell surface heparan sulfate proteoglycans (HSPGs) and involves heparin-binding domains of fibronectin. Woods et al. (1986) EMBO J. 5:665-670.
The formation of complete focal adhesions and stress fibers in the context of integrins has been shown to require integrin clustering, integrin occupancy, tyrosine phosphorylation and cytoskeletal integrity. Hynes (1992) Cell 69:11-25; Clark et al. (1995) Science 268:233-239; Miyamoto et al. (1995) J. Cell Biol. 131:791-805; Miyamoto et al. (1995) Science 267:883-885. Integrin signaling pathways involve the small GTP-binding protein Rho. Parsons (1996) Curr. Opin. Cell Biol. 8:146-152; small GTP-binding protein Rho. Parsons (1996) Curr. Opin. Cell. Biol. 8:146-152; Tapon et al. (1997) Curr. Topics Cell Biol. 9:86-92; Clark et al. (1998) J. Cell Biol. 142:573-586.
The present invention is based, at least in part, on the discovery that syndecan-4 acts cooperatively with integrins in generating signals for cell spreading and for the assembly of focal adhesions and actin stress fibers. In addition, it was discovered that these joint signals are regulated in a Rho-dependant manner.
Specifically, it was found that when a cell, which does not express fibronectin, is plated solely on the cell binding domain of fibronectin or antibodies directed against thexcex2 integrin chain, the cell attaches but fails to spread or assemble focal adhesions or actin stress fibers. When antibodies directed against the ectodomain of syndecan-4 are then added, the cells spread fully and assemble focal adhesions and actin stress fibers which are indistinguishable from cells plated on fibronectin. Moreover, it was found that the activation of the GTP-binding protein Rho, which is a key regulator in adhesion-mediated signaling events through integrins, is also dependent on syndecan-4 signaling events.
Accordingly, in one aspect, the invention features, a method of modulating the spatial or positional relationship of a cell to a substrate, or modulating the intracellular response of a cell to a substrate, in vitro or in vivo. The spatial or positional relationship of a cell to a substrate refers to the spatial or positional relationship of one or more points on the cell to substrate, and includes, e.g., cell attachment, cell spreading, or cell migration. Cell spreading includes the movement of an entire cell across a substrate as well as the movement of one part of the cell but not another part of the cell with regard to the substrate. The intracellular response of a cell to a substrate refers the response, e.g., the movement or formation or disolution of, a cellular component, e.g., a cell membrane protein, or an actin stress fiber, to a substrate and includes the formation or dissolution of a focal adhesion.
The method includes administering an agent which modulates the interaction, e.g., the binding, of the syndecan-4 ectodomain with a counterligand, thereby modulating the spatial or positional relationship of a cell to a substrate, or modulating the intracellular response of a cell to a substrate. The preferred counterligand is an ECM component, e.g., the heparin-binding domain of a component of the extracellular matrix (ECM) such as fibronectin, vitronectin, a laminin or a collagen.
In a preferred embodiment, the agent inhibits syndecan-4 ectodomain binding to a counterligand, e.g., the heparin-binding domain of an ECM molecule. The spatial or positional relationship of a cell to a substrate, or the intracellular response of a cell to a substrate, can be inhibited or reduced by interfering with the interaction of syndecan-4 a counterligand, e.g., a heparin-binding domain of an ECM molecule. Agents which interfere with the binding of syndecan-4 to a counterligand include agents which bind to an ECM molecule, such as fibronectin, vitronectin, a laminin or a collagen, and thereby inhibit the binding of the ectodomain of syndecan-4, with a heparin-binding domain of the ECM molecule. The invention is not limited by the particular mechanism of inhibition. The agent can e.g., act: by binding and occupying the site normally bound by the ectodomain of syndecan-4, by binding to and changing the shape of the ECM molecule (such that counterligand binding is inhibited); or by binding an ECM molecule and sterically hindering binding of the ectodomain of syndecan-4 to a heparin-binding domain of the molecule. Examples of such agents include: a syndecan-4 protein, e.g., a soluble syndecan4 protein, or a heparin binding domain (HBD)-binding portion thereof, e.g., from amino acid residues 90 to 120 of SEQ ID NO:2; a fusion of a syndecan-4 protein, e.g., a fusion of syndecan-4, or a HBD-binding portion thereof, to another polypeptide, e.g., a polypeptide which promotes solubility or which targets or binds to a substrate; a polypeptide other than syndecan-4 which binds to an ECM molecule, e.g., to a heparin binding domain of an ECM molecule, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay; antibodies to an ECM molecule, e.g., antibodies which bind the site normally bound by the ectodomain of syndecan-4, antibodies which bind to and change the shape of the ECM molecule, or antibodies which bind and sterically hinder binding of the ectodomain of syndecan-4 to a heparin-binding domain of an ECM molecule. ECM molecules include, for example, fibronectin, vitronectin, laminins and collagens.
In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
The inhibitory agent can act by binding syndecan-4. While not wishing to be bound by theory these agents may bind to syndecan-4 but fail to provide, or otherwise inhibit the transduction of a signal which normally arises when syndecan-4 binds to a heparin-binding domain of an ECM molecule. These agents can e.g., bind and occupy the site on the ectodomain of syndecan-4 normally bound by a heparin-binding domain of an ECM molecule; bind to and change the shape of syndecan-4; or bind and sterically hinder binding of a heparin-binding domain of an ECM molecule to the ectodomain of syndecan-4. Examples of such agents include an analog of fibronectin or of a syndecan-4 binding portion of fibronectin; an analog of vitronectin or of a syndecan-4 binding portion of vitronectin; an analog of a laminin or of a syndecan-4 binding portion of a laminin; an analog of a collagen or of a syndecan-4 binding portion of a collagen; an analog of a soluble heparin binding domain; a fusion of all, or a syndecan-4 protein binding portion, of an analog of an EMC molecule to another polypeptide, e.g., a polypeptide which promotes solubility or which targets or binds to a substrate; a polypeptide other than a heparin binding domain which binds to the ectodomain of syndecan-4, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay; or an antibody which binds the ectodomain of syndecan-4, e.g., antibodies which bind the ectodomain of syndecan-4 and inhibit the transduction of a signal which normally arises when syndecan-4 binds to a heparin-binding domain of an ECM molecule. These agents can be found by selecting for species which bind to the ectodomain of syndecan-4 but which result in a less than normal level of cell spreading or the formation of focal adhesions. A two part screen can be used wherein binding is selected for in an in vitro or cell free assay and activity is selected for in a cell based assay. In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
In another preferred embodiment, the agent promotes cell spreading, cell attachment, cell migration, or the formation of a focal adhesion. While not wishing to be bound by theory, these agents are believed to act by mimicking (or agonizing) the binding of a heparin binding domain of an ECM molecule such as fibronectin to the ectodomain of syndecan-4. These agents can bind the site on the ectodomain of syndecan-4 normally bound by a heparin-binding domain of an ECM molecule. Examples of such agents include: fibronectin, or a syndecan-4 binding portion of fibronectin; vitronectin, or a syndecan-4 binding portion of vitronectin; a laminin, or a syndecan-4 binding portion of a laminin; a collagen, or a syndecan-4 binding portion of a collagen; a soluble a heparin binding domain; a fusion of all or a syndecan-4 protein binding portion of an ECM molecule to another polypeptide, e.g., a polypeptide which promotes solubility or which targets or binds to a substrate; a polypeptide other than a heparin binding domain which binds to the ectodomain of syndecan-4, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay; or an antibody which binds the ectodomain of syndecan-4. These agents can be found by selecting for species which bind to the ectodomain of syndecan-4 and which result in cell spreading or the formation of focal adhesions. A two part screen can be used wherein binding is selected for in an in vitro or cell free assay and activity is selected for in a cell based assay. In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
In a preferred embodiment, the method of promoting cell attachment, cell spreading, migration, or the formation of a focal adhesion, further comprises administering an agent which promotes integrin binding, e.g., an anti-integrin antibody. Preferably, the antibody is an anti-xcex21 integrin chain antibody. Other agents which promote integrin binding include: a fibronectin protein, e.g., the cell binding domain (CBD) portion of fibronectin, e.g., the RGD sequence of the CBD; a vitronectin protein, e.g., the CBD portion of vitronectin; a laminin protein, e.g., the CBD portion of a laminin; a collagen protein, e.g., the CBD portion of a collagen; a polypeptide other than an ECM molecule which binds to a xcex21 chain of integrin, e.g., a polypeptide which binds to a CBD-binding portion of integrin, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay; a small molecule, e.g., a small molecule capable of binding a xcex21 chain of integrin, e.g., a small molecule capable of binding a CBD-binding portion of integrin. In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
In a preferred embodiment, the method modulates, e.g., inhibits or promotes, cell attachment and/or spreading by: modulating focal adhesion assembly; modulating actin stress fiber formation; modulating an adhesion-mediated signaling pathway, e.g., modulating a Rho-dependent adhesion pathway; modulating the interaction, e.g., binding, with a component of the extracellular matrix, e.g., fibronectin, vitronectin, a laminin, or a collagen.
In a preferred embodiment, the method includes treating a subject having a disorder characterized by unwanted or abnormal cellular interactions (e.g., unwanted or abnormal cell-cell and/or cell-matrix interactions, unwanted or abnormal cell migration/movement, e.g., cancer).
In another aspect, the invention features a method of modulating the spatial or positional relationship of a cell to a substrate, or modulating the intracellular response of a cell to a substrate.
In a preferred embodiment, the invention features a method of inhibiting syndecan-4 ectodomain binding to a counterligand, e.g., the heparin binding domain of an ECM molecule, and an agent which inhibits integrin binding. Agents which interfere with the binding of syndecan-4 to a counterligand include agents which bind to an ECM molecule (e.g., fibronectin, vitronectin, a laminin, or a collagen) and thereby inhibit the binding of the ectodomain of syndecan-4, with a heparin-binding domain of the ECM molecule. The invention is not limited by the particular mechanism of inhibition. The agent can, e.g., act: by binding and occupying the site normally bound by the ectodomain of syndecan-4, by binding to and changing the shape of the ECM molecule (such that counterligand binding is inhibited); or by binding an ECM molecule and sterically hindering binding of the ectodomain of syndecan-4 to a heparin-binding domain of the ECM molecule. Examples of such agents include: a syndecan-4 protein, e.g., a soluble syndecan-4 protein, or a HBD-binding portion thereof e.g., from about amino acids 90 to 120 of SEQ ID NO:2; a fusion of a syndecan-4 protein, e.g., a fusion of syndecan-4, or a HBD-binding portion thereof, to another polypeptide, e.g., a polypeptide which promotes solubility or which targets or binds to a substrate; a polypeptide other than syndecan-4 which binds to an ECM molecule e.g., to a heparin binding domain of an ECM molecule, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay; antibodies to an ECM molecule, e.g., antibodies which bind the site normally bound by the ectodomain of syndecan-4, antibodies which bind to and change the shape of the ECM molecule, or antibodies which bind and sterically hinder binding of the ectodomain of syndecan-4 to a heparin-binding domain of an ECM molecule.
In a preferred embodiment, the agent which inhibits or reduces integrin binding is: an integrin protein, e.g., a soluble integrin protein, or a CBD-binding portion thereof; a fusion of an integrin protein, e.g., a fusion of a integrin, or a CBD-binding portion thereof, to another polypeptide, e.g., a polypeptide which promotes solubility; a polypeptide other than integrin which binds to a cell binding domain of fibronectin, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay; antibodies to an ECM molecule, e.g., antibodies which bind to the site normally bound by integrin, antibodies which bind to and change the shape of the ECM molecule, or antibodies which bind and sterically hinder binding of the cell binding domain-binding portion of integrin. In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
The inhibitory agent can act by binding syndecan-4. While not wishing to be bound by theory these agents may bind to syndecan-4 but fail to provide, or otherwise inhibit the transduction of a signal which normally arises when syndecan-4 binds to a heparin-binding domain of an ECM molecule. These agents can e.g., bind and occupy the site on the ectodomain of syndecan-4 normally bound by a heparin-binding domain of an ECM molecule; bind to and change the shape of syndecan-4; or bind and sterically hinder binding of a heparin-binding domain of an ECM molecule to the ectodomain of syndecan-4. Examples of such agents include an analog of fibronectin or of a syndecan-4 binding portion of fibronectin; an analog of vitronectin or of a syndecan-4 binding portion of vitronectin; an analog of a laminin or of a syndecan-4 binding portion of a laminin; an analog of a collagen or of a syndecan-4 binding portion of a collagen; an analog of a soluble heparin binding domain; a fusion of all, or a syndecan-4 protein binding portion, of an analog of an ECM molecule to another polypeptide, e.g., a polypeptide which promotes solubility or which targets or binds to a substrate; a polypeptide other than a heparin binding domain which binds to the ectodomain of syndecan-4, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay; or an antibody which binds the ectodomain of syndecan-4, e.g., antibodies which bind the ectodomain of syndecan-4 and inhibit the transduction of a signal which normally arises when syndecan-4 binds to a heparin-binding domain of an ECM molecule. These agents can be found by selecting for species which bind to the ectodomain of syndecan-4 but which result in a less than normal level of cell spreading or the formation of focal adhesions. A two part screen can be used wherein binding is selected for in an in vitro or cell free assay and activity is selected for in a cell based assay. In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
In another preferred embodiment, the invention features a method of promoting cell attachment, cell spreading, migration, or the formation of a focal adhesion. The method includes administering an agent which promotes syndecan-4 ectodomain binding and an agent which promotes integrin binding. Agents which promote syndecan-4 ectodomain binding can bind the site on the ectodomain of syndecan-4 normally bound by a heparin-binding domain of an ECM molecule. Examples of such agents include: fibronectin, or a syndecan-4 binding portion of fibronectin; vitronectin, or a syndecan-4 binding portion of vitronectin; a laminin, or a syndecan-4 binding portion of a laminin; a collagen, or a syndecan-4 binding portion of a collagen; a soluble a heparin binding domain; a fusion of all or a syndecan-4 binding portion of an ECM molecule to another polypeptide, e.g., a polypeptide which promotes solubility; a polypeptide other than a heparin binding domain which binds to the ectodomain of syndecan-4, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay; or an antibody which binds the ectodomain of syndecan-4. These agents can be found by selecting for species which bind to the ectodomain of syndecan-4 and which result cell spreading or the formation of focal adhesions. In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
In a preferred embodiment, the agent which promotes integrin binding is: an anti-integrin antibody, e.g., an anti-xcex21 integrin chain antibody; a fibronectin protein, e.g., the CBD portion of fibronectin, e.g., the RGD sequence of the CBD; a vitronectin, or an integrin binding portion of vitronectin; a laminin, or an integrin binding portion of a laminin; a collagen, or an integrin binding portion of a collagen; a polypeptide other than an ECM molecule which binds to a xcex21 chain of integrin, e.g., a CBD-binding portion of integrin, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay; a small molecule, e.g., a small molecule capable of binding a xcex21 chain of integrin, e.g., a CBD-binding portion of integrin. In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
In a preferred embodiment, the method modulates, e.g., inhibits or promotes, cell attachment and/or spreading by: modulating focal adhesion assembly; modulating actin stress fiber formation; modulating an adhesion-mediated signaling pathway e.g., a Rho dependent adhesion pathway; modulating the interaction, e.g., binding, with a component of the extracellular matrix, e.g., fibronectin, vitronectin, a laminin, a collagen.
In a preferred embodiment, the method includes treating a subject having a disorder characterized by unwanted or abnormal cellular interactions (e.g., unwanted or abnormal cell-cell and/or cell-matrix interactions, unwanted or abnormal cell migration/movement, e.g., cancer).
Another aspect of the invention features a method of identifying a compound or agent which modulates the spatial or positional relationship of a cell to a substrate, or modulating the intracellular response of a cell to a substrate.
In a preferred embodiment, the compound inhibits or reduces cell attachment, cell spreading, migration or the formation of a focal adhesion. A compound can include, for example, a fragment or analog of syndecan-4, a polypeptide other than syndecan-4, e.g., a randomly generated polypeptide which interacts with an ECM molecule, e.g., interacts with the heparin binding domain of an ECM molecule; or a small molecule, e.g., a small molecule which interacts with an ECM molecule, e.g., interacts with the heparin binding domain of an ECM molecule. In a preferred embodiment, the method can include the steps of forming a reaction mixture which includes a polypeptide comprising the heparin binding domain of an ECM molecule and a compound or agent under conditions which allow for the binding of the compound to the heparin binding domain to form a complex and detecting the formation of a complex of the heparin binding domain and the compound, in which the ability of the compound to bind the heparin binding domain is indicated by the presence of the compound in complex.
Methods for identifying a compound or an agent can be performed, for example, using a cell free assay. For example, the heparin binding domain of an ECM molecule such as fibronectin can be immobilized to a suitable substrate, e.g., glutathoine sepharose beads or glutathoine derivatized microtitre plates, using a fusion protein which allows for the heparin binding domain to bind to the substrate, e.g., a glutathoine-S-transferase/heparin binding domain fusion protein.
In a preferred embodiment, the method further comprises adding syndecan-4, or a polypeptide comprising the heparin binding domain-binding region of syndecan-4, to the reaction mixture.
In a preferred embodiment, a compound can be identified using a cell-based assay. These methods include identifying a compound based on its ability to modulate, e.g., inhibit, an adhesion-mediated activity of the cell. Example of such adhesion mediated activities include: inhibiting focal adhesion assembly, inhibiting actin stress fiber formation; inhibiting an adhesion-mediated signaling pathway, e.g., a Rho dependent signaling pathway; inhibiting the interaction, e.g., binding, with a component of the extracellular matrix, e.g., fibronectin, vitronectin, a laminin, or a collagen.
In another preferred embodiment, the compound promotes or increases cell attachment, cell spreading, migration or formation of a focal adhesion. A compound can include, for example, an analog of fibronectin, e.g., an analog of the heparin binding domain; an analog of vitronectin, or a syndecan-4 binding portion of vitronectin; an analog of a laminin, or a syndecan-4 binding portion of a laminin; an analog of a collagen, or a syndecan-4 binding portion of a collagen; a polypeptide other than an EMC molecule, e.g., a randomly generated polypeptide which interacts with the syndecan-4, e.g., the HBD-binding portion of syndecan-4; or a small molecule, e.g., a small molecule which interacts with syndecan-4, e.g., the ectodomain of syndecan-4, e.g., the HBD-binding portion of syndecan-4. In a preferred embodiment, the method can include the steps of forming a reaction mixture which includes a polypeptide comprising the HBD-binding portion of syndecan-4, with a compound or agent under conditions which allow for the binding of the compound to the HBD-binding portion to form a complex and detecting the formation of a complex of the HBD-binding portion and the compound, in which the ability of the compound to bind the HBD-binding portion of syndecan-4 is indicated by the presence of the compound in complex.
Methods for identifying a compound or an agent can be performed, for example, using a cell free assay. For example, the HBD-binding portion of syndecan-4 can be immobilized to a suitable substrate, e.g., glutathoine sepharose beads or glutathoine derivatized microtitre plates, using a fusion protein which allows for the HBD-binding portion to bind to the substrate, e.g., a glutathoine-S-transferase/HBD-binding portion of syndecan-4 fusion protein.
In a preferred embodiment, a compound can be identified using a cell based assay. These methods include identifying a compound based on its ability to modulate, e.g., promote, an adhesion-mediated activity of the cell. Example of such adhesion mediated activities include: promoting focal adhesion assembly, promoting actin stress fiber formation; promoting an adhesion-mediated signaling pathway, e.g., a Rho dependent signaling pathway; promoting the interaction, e.g., binding, with a component of the extracellular matrix, e.g., fibronectin, vitronectin, a laminin, a collagen.
In another aspect, the invention features a method of identifying a compound which modulates, e.g., inhibits or promotes, the interaction of syndecan-4 ectodomain with an agent which binds to syndecan4, e.g., a heparin binding domain of an ECM molecule. ECM molecules include fibronectin, vitronectin, laminins, and collagen. In these methods, a polypeptide comprising the heparin binding domain of an ECM molecule is contacted, in the presence a compound or agent, with syndecan-4, or a portion thereof, under conditions which allow binding of the heparin binding domain and syndecan-4. An alteration, e.g., an increase or decrease, in complex formation between the heparin binding domain and syndecan-4, as compared to the amount formed in the absence of the compound or agent is indicative of ability of the compound to modulate the interaction of the heparin binding domain and syndecan-4.
Another aspect of the invention features a method of identifying a compound which modulates, inhibits or reduces, cell attachment and/or spreading. The method includes contacting a polypeptide comprising the heparin binding domain of an ECM molecule with a compound or agent in the presence of a cell which expresses syndecan-4, or a portion thereof, and detecting the presence or absence of an adhesion-mediated activity by the cell. These methods can include detecting the following adhesion mediated activities: modulation of focal adhesion assembly; modulation actin stress fiber formation; modulation an adhesion-mediated signaling pathway, e.g., a Rho-dependent signaling pathway; modulation the interaction, e.g., binding with a component of the extracellular matrix, e.g., fibronectin, vitronectin, a laminin, a collagen. Such compounds can include, for example, a fragment or analog of syndecan-4, a polypeptide other than syndecan-4, e.g., a randomly generated polypeptide which interacts with an ECM molecule, e.g., the heparin binding domain of an ECM molecule; or a small molecule, e.g., a small molecule which interacts with an ECM molecule, e.g., a small molecule which interacts with the heparin binding domain of an ECM molecule. In addition, a compound can bind to syndecan-4, e.g., the ectodomain of syndecan-4, without generating adhesion-mediated signals. Such compounds can include, for example, an analog of a heparin binding domain which binds to syndecan-4 but does not generate adhesion-mediated signaling; a polypeptide other than a heparin binding domain, e.g., a randomly generated polypeptide which does not bind the HBD-binding portion of syndecan-4, but blocks binding of other agents to the HBD-binding portion of syndecan-4, or a randomly generated polypeptide which binds to the HBD-binding portion of syndecan-4 but does not generate adhesion-mediated signaling; or a small molecule, e.g., a small molecule which does not bind the HBD-binding portion of syndecan-4, but blocks binding of other agents to the HBD-binding portion of syndecan-4, or a small molecule which binds to the HBD-binding portion of syndecan-4 but does not generate adhesion-mediated signaling.
In another aspect, the invention features, a method of treating a subject having a disorder characterized by unwanted or abnormal cell adhesion or cell spreading (e.g., cancer). The method includes administering to the subject an agent which modulates, e.g., inhibits, the binding of syndecan-4 with a couterligand, thereby modulating, e.g., inhibiting, cell adhesion or cell spreading.
In a preferred embodiment, the agent inhibits syndecan4 ectodomain binding to a counterligand, e.g., an ECM molecule, e.g., the heparin-binding domain of an ECM molecule. The spatial or positional relationship of a cell to a substrate, or the intracellular response of a cell to a substrate, can be inhibited or reduced by interfering with the interaction of syndecan4 a counterligand, e.g., a heparin-binding domain of an ECM molecule. Examples of ECM molecules include fibronectin, vitronectin, laminins and collagen. Agents which interfere with the binding of syndecan-4 to a counterligand include agents which bind to an ECM molecule, e.g., a heparin binding domain of an ECM molecule, and thereby inhibit the binding of the ectodomain of syndecan-4, with a heparin-binding domain of the ECM molecule. The invention is not limited by the particular mechanism of inhibition. The agent can e.g., act: by binding and occupying the site normally bound by the ectodomain of syndecan-4, by binding to and changing the shape of the ECM molecule (such that counterligand binding is inhibited); or by binding an ECM molecule and sterically hindering binding of the ectodomain of syndecan4 to a heparin-binding domain of an ECM molecule. Examples of such agents include: a syndecan-4 protein, e.g., a soluble syndecan-4 protein, or a HBD-binding portion thereof e.g., from about amino acids 90 to 120 of SEQ ID NO:2; a fusion of a syndecan-4 protein, e.g., a fusion of syndecan-4, or a HBD-binding portion thereof, to another polypeptide, e.g., polypeptide which promotes solubility or which targets or binds to a substrate; a polypeptide other than syndecan-4 which binds to an ECM molecule, e.g., to a heparin binding domain of an ECM molecule, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay; antibodies to an ECM molecule, e.g., antibodies which bind the site normally bound by the ectodomain of syndecan-4, antibodies which bind to and change the shape of the ECM molecule, or antibodies which bind and sterically hinder binding of the ectodomain of syndecan-4 to a heparin-binding domain of an ECM molecule.
In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
The inhibitory agent can act by binding syndecan-4. While not wishing to be bound by theory these agents may bind to syndecan-4 but fail to provide, or otherwise inhibit the transduction of a signal which normally arises when syndecan-4 binds to a heparin-binding domain of an ECM molecule, e.g., fibronectin, vitronectin, a laminin, or a collagen. These agents can e.g., bind and occupy the site on the ectodomain of syndecan-4 normally bound by a heparin-binding domain of an ECM molecule; bind to and change the shape of syndecan-4; or bind and sterically hinder binding of a heparin-binding domain of an ECM molecule to the ectodomain of syndecan-4. Examples of such agents include an analog of fibronectin, or a syndecan-4 binding portion of fibronectin; an analog of vitronectin, or a syndecan-4 binding portion of vitronectin; an analog of a laminin, or a syndecan4 binding portion of a laminin; an analog of a collagen, or a syndecan-4 binding portion of a collagen; an analog of a soluble heparin binding domain of an ECM molecule; a fusion of all, or a syndecan-4 protein binding portion, of an ECM molecule to another polypeptide, e.g., a polypeptide which promotes solubility or which targets or binds to a substrate; a polypeptide other than a heparin binding domain which binds to the ectodomain of syndecan-4, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay; or an antibody which binds the ectodomain of syndecan-4. These agents can be found by selecting for species which bind to the ectodomain of syndecan-4 but which result in a less than normal level of cell spreading or the formation of focal adhesions. A two part screen can be used wherein binding is selected for in an in vitro or cell free assay and activity is selected for in a cell based assay. In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
In a preferred embodiment, the method further comprises administering an agent which inhibits integrin binding. Agents which inhibit or reduce integrin binding include: an integrin protein, e.g., a soluble integrin protein, or a CBD-binding portion thereof; a fusion of an integrin protein, e.g., a fusion of a integrin, or a CBD-binding portion thereof, to another polypeptide, e.g., a polypeptide which promotes solubility; a polypeptide other than integrin which binds to a cell binding domain of an ECM molecule, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay; antibodies to an ECM molecule, e.g., antibodies which bind to the site normally bound by integrin, antibodies which bind to and change the shape of an ECM molecule, or antibodies which bind and sterically hinder binding of the cell binding domain-binding portion of integrin. In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
In a preferred embodiment, the method modulates, e.g., inhibits, cell attachment and/or spreading by: modulating focal adhesion assembly; modulating actin stress fiber formation; modulating an adhesion-mediated signaling pathway, e.g., modulating a Rho-dependent adhesion pathway; modulating the interaction, e.g., binding, with a component of the extracellular matrix, e.g., fibronectin, vitronectin, a laminin, a collagen.
Another aspect of the invention features a method of treating a subject having a disorder characterized by abnormal cell adhesion or cell spreading. The method includes administering to the subject an agent which modulates, e.g., promotes, the binding of syndecan-4 with a couterligand, thereby modulating, e.g., promoting, cell adhesion or cell spreading.
In a preferred embodiment, the agent promotes cell spreading, cell attachment, cell migration, or the formation of a focal adhesion. While not wishing to be bound by theory, these agents are believed to act by mimicking (or agonizing) the binding of a heparin binding domain of an ECM molecule to the ectodomain of syndecan-4. These agents can bind the site on the ectodomain of syndecan-4 normally bound by a heparin-binding domain of an ECM molecule. Examples of such agents include: fibronectin, or a syndecan-4 binding portion of fibronectin; vitronectin, or a syndecan-4 binding portion of vitronectin; a laminin, or a syndecan-4 binding portion of a laminin; a collagen, or a syndecan-4 binding portion of a collagen; a soluble a heparin binding domain of an ECM molecule; a fusion of all or a syndecan-4 protein binding portion of an ECM molecule to another polypeptide, e.g., a polypeptide which promotes solubility or which targets or binds to a substrate; a polypeptide other than a heparin binding domain which binds to the ectodomain of syndecan-4, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay; or an antibody which binds the ectodomain of syndecan-4. These agents can be found by selecting for species which bind to the ectodomain of syndecan-4 and which result in cell spreading or the formation of focal adhesions. A two part screen can be used wherein binding is selected for in an in vitro or cell free assay and activity is selected for in a cell based assay. In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
In a preferred embodiment, the method of promoting cell attachment, cell spreading, migration, or the formation of a focal adhesion, further comprises administering an agent which promotes integrin binding, e.g., an anti-integrin antibody. Preferably, the antibody is an anti-xcex21 chain antibody. Other agents which promote integrin binding include: a fibronectin protein, e.g., the CBD portion of fibronectin, e.g., the RGD sequence of the CBD; a vitronectin, e.g., a CBD portion of vitronectin; a laminin, or a CBD portion of a laminin; a collagen, or a CBD of a collagen; a polypeptide other than an ECM molecule which binds to a xcex21 chain of integrin, e.g., a polypeptide which binds to a CBD-binding portion of integrin, e.g., a polypeptide selected for binding in, e.g., a phage display or 2 hybrid assay; a small molecule, e.g., a small molecule capable of binding a xcex21 chain of integrin, e.g., a small molecule capable of binding a CBD-binding portion of integrin. In a preferred embodiment, the method includes administering a nucleic acid which encodes one of the above-described agents.
In a preferred embodiment, the method modulates, e.g., promotes, cell attachment and/or spreading by: modulating focal adhesion assembly; modulating actin stress fiber formation; modulating an adhesion-mediated signaling pathway, e.g., modulating a Rho-dependent adhesion pathway; modulating the interaction, e.g., binding, with a component of the extracellular matrix, e.g., fibronectin, vitronectin, a laminin, a collagen.
The term xe2x80x9ctreatmentxe2x80x9d, as used herein, refers to any therapeutic treatment, e.g., the administration of a therapeutic agent or substance, e.g., a drug.
As used herein, the term xe2x80x9csubjectxe2x80x9d refers to human and non-human animals. In preferred embodiments, the subject is a human, e.g., a person having or diagnosed as at risk for a disorder characterized by aberrant or unwanted cell attachment and/or spreading. The term xe2x80x9cnon-human animalsxe2x80x9d of the invention includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, ruminants, birds, amphibians, and reptiles.
The terms xe2x80x9cpeptidesxe2x80x9d, xe2x80x9cproteinsxe2x80x9d and xe2x80x9cpolypeptidesxe2x80x9d are used interchangeably herein.
The term xe2x80x9csmall moleculesxe2x80x9d as used herein refers, to a molecule which has a molecular weight of less than 2,000, preferably less than 1,000. Examples of small molecules include peptides, peptidomimitics, and non-peptidic compounds such as organic molecules. Such molecules can bind to an ECM molecule, e.g., the HBD of an ECM molecule, to inhibit or reduce syndecan-4 binding or can bind to syndecan-4 to either inhibit or reduce the binding of syndecan-4 to an ECM molecule, e.g., HBD, or can bind the syndecan-4 to promote cell adhesion.
The term xe2x80x9cectodomainxe2x80x9d refers to the extracellular portion of sydecan-4.
An xe2x80x9cadhesion mediated activityxe2x80x9d, as used herein, refers to activities of a cell which modulate the spatial or positional relationship of a cell to a substrate or modulates the intracellular response of a cell to a substrate. The xe2x80x9cspatial or positional relationship of a cell to a substratexe2x80x9d refers to the spatial or positional relationship of one or more points on the cell to substrate, and includes e.g., cell attachment, cell spreading, or cell migration. Cell spreading includes the movement of an entire cell across a substrate as well as the movement of one part of the cell but not another part of the cell with regard to the substrate. The xe2x80x9cintracellular response of a cell to a substratexe2x80x9d refers the response, e.g., the movement or formation or disolution of, a cellular component, e.g., a cell membrane protein, or an actin stress fiber, to a substrate and includes the formation or dissolution of a focal adhesion. Responses of the cell can also include activation of various intracellular proteins.
The term xe2x80x9csubstratexe2x80x9d as used herein refers to a substrate on which cells can adhere, spread, migrate or form a focal adhesion either in vivo, or in vitro. In vivo examples include a basement membrane, a layer of cells, or the surface of an organ or tissue. Generally this is mediated by a counterligand on or in the substrate. A counterligand can be a component of the ECM, e.g., components of the extracellular matrix such as fibronectin, vitronectin, laminins, and collagens, as well as components of other cells which provide for attachment.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are described in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames and S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames and S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.